This novel SERS platform exhibits high SERS performance in terms of sensitivity, reproducibility and practicability mainly because there are high-density and multi-level “hot spots” in the appropriate nanogaps. Meanwhile, it also systematically compares the differences of the SERS performance of Cu and Ag decorated M.w. hybrids and how these differences can alter their response. Moreover, the proposed substrate is employed to rapidly detect the pesticide residues on apple peels and the LOD for cypermethrin is estimated at 10−10 mg/mL. Therefore, this novel SERS substrate has great potential in rapid sampling of pesticide residues on real samples and expands the investigation to other natural materials for fabricating various SERS platforms.The recent development of SERS substrates based on irregular nanostructures for directly molecule recognition has aroused increasing attention. By combining the irregular flake-like nanostructures of mantis wings, high SERS performance of Ag nanofilms, and the chemical stability of Au nanoparticles (NPs), an ultra-sensitive and flexible SERS substrate based on Au NPs functionalized Ag nanofilms-mantis wings (Au-Ag-M.w.) hybrid system is successfully fabricated. When 4-aminothiophenol is selected as the probe molecule, the limit of detection (LOD) is as low as 10 Raman scattering spectroscopy is a rapid, nondestructive and qualitative analysis tool which can provide vibrational and rotational information of a molecule . Recent |E|4 .In general, coinage metals such as Au, Ag and Cu with specific plasmonic nanostructures are the most popular SERS platforms ,14,15,16−9 M [−11 M for malachite green) and well reproducibility of Raman signals were due to the super-hydrophobicity and flexible surface. That is to say the SERS performance can be enhanced by the hydrophobic surface. Specifically, when a droplet of probe molecule solution is placed on the hydrophobic nanomaterial, the droplet will form to be a near-spherical shape. After evaporation, the probe molecules are highly concentrated into a tiny area, resulting in high sensitivity. In addition to high sensitivity, excellent Raman enhancement and super-hydrophobicity, these biomaterial-based SERS substrates possess another advantage in flexibility. They offer advantages over traditional rigid substrates in their flexibility and can sense analytes on the surface of non-planar geometries. What’s more, they possess excellent mechanical strain resistance and can be easily tailored into any desired shapes and sizes. This point has been well demonstrated and analyzed by many reported flexible SERS substrates [Recently, plenty of scientific researches have confirmed that the natural world possesses multifunctional nanomaterials and nanostructures which can be applied in SERS applications due to the unique surface characteristics and low-cost acquirement of biomaterials. Meanwhile, considerable research efforts have suggested that simply modified with noble metals, biomaterials such as insect wings and plant leaves show excellent SERS effect for simultaneous determination of multi-molecules. For example, Mu et al. reported a novel SERS platform based on Au NPs/butterfly wing  as flexible and adhesive SERS substrate. The high-performance SERS substrate was employed to detect 4-aminothiophenol and the LOD was as low as 10−9 M . Accordi−9 M . By usin−9 M . The resbstrates ,32,33.Fascinated by the flexible and hydrophobic bio-materials, we selected M.w. nanoarrays as bio-templates to fabricate an Au/Ag-decorated natural SERS substrate. As shown in 3·HCl·4H2O (99.8%) and Na3C6H5O7·2H2O (99.9%) were purchased from Tianjin Meizke Chemical Co., Ltd. . Deionized water (18.25 MΩ) was used for all solution preparations. Silver target (99.99%) was obtained from China Material Technology Co., Ltd.  M.w.s  were purchased from Beijing Jiaying Grand Life Sciences Co., Ltd. . Experiments with mantis wings complied with the accepted ethical standards and were approved by the Ethical Review Board of Yanshan University on 15 January 2018. R6G, 4-aminothiophenol and cypermethrin were obtained from J&K Scientific Ltd. . AuClAg nanofilms were coated on the M.w.s by the DC magnetron sputtering system (DHRM-3) . The surface morphology and size distribution of different substrates were characterized by field emission scanning electron microscopy (FE-SEM) (S-4800) . The morphology and size of Au NPs were characterized by Transmission Electron Microscope (TEM) (HT7700) . Raman spectra of 4-aminothiophenol, R6G and cypermethrin were obtained by Raman system (inVia) . The UV-vis absorption spectra were monitored by the UV-2550 UV-vis spectrophotometer (Shimadzu (China) Co., Ltd., Shanghai, China).2 and then cleansed by ethanol and deionized water for 10 min in turn to remove the organic contaminants. After natural drying, the Ag nanofilms were deposited. Sputtering process was carried in Argon gas with 99.9% purity and the vacuum degree of sputtering chamber was controlled at 2 × 10−2 mbar. The sputtering voltage was 90 V, the working current 170 mA. Au NPs were prepared by chemical reduction method according to modified Frens’s work [x min would be called Ag-M.w.-x substrate. Consistently, the Au-Ag-M.w.-x described these Ag-M.w.-x substrates decorated with Au NPs.The M.w.s were dissected into approximately 1 × 1 cms’s work . The Au −1. Meanwhile, the power of the laser was set at 0.1 mW to avoid structural changes due to laser heating. The diffraction grating was used 1800 lines/mm grating and the focal length was 250 mm. A 4-aminothiophenol stock solution of the concentration of 0.1 M has been used for further dilution from 10−2 M to 10−13 M. The 4-aminothiophenol aqueous solution (10 µL) was dropped on each fabricated SERS substrate prior to the SERS measurement, and dried in a vacuum drying oven at 40 °C for 5 min. The Raman spectra were recorded with an integrated accumulation time of 10 s. When dealing with the Raman data, a background removal algorithm was adopted, and the Raman spectra were smoothed to remove the disorderly peaks. In order to obtain a statistical result and reduce the influence caused by the laser, the Raman spectra were taken at five different areas of each sample and the collected Raman data were averaged.All the Raman spectra were obtained with a ×50 objective lens (NA = 0.75). For SERS measurements, the laser with the wavelength of 532 nm was used as the excitation source. The diameter of the laser spot was ~1 μm and the spectral resolution was about 1 cm2. Subsequently, 10 μL different concentrations of cypermethrin solutions were separately dropped onto the peel samples with a pipette. After natural drying, 10 μL ethanol solution was added onto the peels to extract the pesticide residues and increase the concentration of cypermethrin molecules at the outer surface. The next step was to press the Au-Ag-M.w.-20 substrate to the treated sites and remained until completely dry and then peeled off for further analysis. In order to ensure the successful acquisition of Raman signals of cypermethrin, the measurements were performed five times randomly on the treated peels and the collected Raman data were averaged.The trace detection of cypermethrin on apple peels was followed Liu et al.’s method . First, x substrates, 4-aminothiophenol, which has been widely applied in SERS detection, was chosen as the probe molecule due to its well established vibrational features. −4 M 4-aminothiophenol recorded from different Ag-M.w. substrates. The strong characteristic peaks of 4-aminothiophenol on the Ag-M.w.-x substrates were mainly centered at 1007, 1077, 1140, 1180, 1367, 1453, 1578 and 1650 cm−1 and the characteristic vibrational modes were given in x to realize the coupling effect of two kinds of localized electromagnetic fields between Ag nanofilms and Au NPs. Hence, Au NPs with the average diameter of 30 ± 2 nm were deposited on the Ag-M.w.-x substrates to fabricate the Au-Ag-M.w.-x substrates. As shown in −4 M 4-aminothiophenol exhibited higher enhancement than those of Ag-M.w.-x hybrids. More importantly, the trend of the Raman signal enhancement was in agreement with that of Ag-M.w.-x. As shown in x substrates was much higher than those of the Ag-M.w.-x substrates, especially for Au-Ag-M.w.-20 substrate. This active result can be attributed to the successful assembly of AuNPs on the Ag nanofilm. And the SERS substrate obtained the high enhancement with the assist of Au NPs which created high-density electromagnetic “hot spots”. Compared with the optimal SERS substrate (Cu-M.w.-30) in the previous report, the SERS substrates in our work is better than that one in terms of SERS enhancement. The SERS spectra of 10−4 M 4-aminothiophenol adsorbed onto Ag-M.w.-20 substrate, Au-Ag-M.w.-20 substrate, Cu-M.w.-30 substrate as well as 4-aminothiophenol solid powder were all exhibited in −1 for example, as shown in In our previous report , we haveThe prepared flexible substrates were characterized by FE-SEM. As shown in −1 was obtained when the angle was 45°. In this case, the calculated EF was evaluated to be 1.49 × 107. Meanwhile, the model and the distribution of electromagnetic field intensity of Cu-M.w.-30 and Ag-M.w.-20 nanofilms with the angles crossed at 45° was also shown in 4) and Ag-M.w.-20 (EF = 8.07 × 105) were weaker than Au-Ag-M.w.-20 SERS-active substrates. Simulation analysis shows that the types of noble metal material, size and nanofilm separation were crucial to efficiently tune the SERS performance. On the other hand, the novel flake-like nanostructure and the synergistic effects of Ag nanofilms and Au NPs would all contribute to the major enhancement of the electromagnetic field.Except for the rich collection effect resulted from the surface hydrophobicity, the types of noble metal materials and the LSPR effect excited near the nanostructure surface could also contribute to the electromagnetic field intensity . Therefo−9 M to 10−13 M). For comparison, the sensitivity of Ag-M.w.-20 substrate was also determined. As shown in −13 M. The LOD for Au-Ag-M.w.-20 substrate was two orders of magnitude lower than that of the Ag-M.w.-20 substrate. It indicated that the sensitivity of Au-Ag-M.w.-20 substrate was clearly superior to the Ag-M.w.-20 hybrid which allowed highly sensitive SERS detection. The better SERS behavior of Au-Ag-M.w.-20 substrate can be attributed to the successful decoration of Au NPs. After decorated the Ag nanofilms with Au NPs, the little probe molecules solution could certainly flow into the tiny nanogaps and could be more enriched around the “hot spots”, thus, generating more sensitive SERS signals. Meanwhile, in our previous report, the LOD for 4-aminothiophenol of Cu-M.w.-30 substrate was 10−6 M as shown in −1 and the 4-aminothiophenol concentration were plotted in the logarithm scale in −1 of 4-aminothiophenol recorded by Cu-M.w.-30 substrates. The values of the correlation coefficient (R2) reached 0.988, 0.979 and 0.976 suggested that these types of substrates could all allow for the quantitative determination of the unknown concentrations of 4-aminothiophenol, but there is no doubt that the Au-Ag-M.w.-20 is the best candidate.To further investigate the SERS sensitivity of the Au-Ag-M.w.-20 substrate, we tested the Raman spectra of 4-aminothiophenol solutions with different concentrations  [n is 25 in agreement with the number of the measured spectrum, −1. Meanwhile, the substrate-to-substrate reproducibility was also measured at 25 randomly chosen points from 5 Au-Ag-M.w.-20 substrates. As shown in −1 were calculated to be 5.53%, 7.15% and 5.93%, as shown in −5 M. The Raman spectra of R6G from Au-Ag-M.w.-20 substrate for different detection times were displayed in −1, the Raman signal intensity fell only by 10.59% within 30 days. In consequence, our Au-Ag-M.w.-20 substrate possesses outstanding reproducibility well antioxidant stability compared to Cu-M.w.-30 substrate [In addition to the high sensitivity, the reproducibility of the Raman signals is a crucial parameter which affects the reliability in application. In general, the RSD value of the Raman signal intensity is usually calculated to evaluate the reproducibility of a SERS substrate. It has been widely confirmed that a RSD value of the SERS measurement less than 20% over micrometer-sized area indicated high reproducibility . Therefotion (1) :(1)RSD=∑ubstrate  which caSERSI and RamanI represent the integrated peak intensity of the SERS signal and the normal Raman signal, SERSN and RamanN are the number of the probe molecules on the substrates within the laser spot. Here, we chose the 10−3 M 4-aminothiophenol solution on the silicon wafer as the Raman substrate, while SERS substrate was used the Au-Ag-M.w.-20 hybrid with 10−9 M 4-aminothiophenol. Besides, the Raman characteristic band of 1367 cm−1 was chosen to calculate the integrated I values. As shown in 1,b2, the value of SERSI and RamanI calculated to be 3.40 × 105 and 8.56 × 104, respectively. Therefore, the ratio SERSI/RamanI was equaled to ~3.98. The RamanN and SERSN can be calculated as followed [AN and M are the Avogadro constant and molar concentration of the solution, solutionV stands for the volume of the droplet of 4-aminothiophenol, subS denotes the size of the 4-aminothiophenol solution on the different substrates and laserS is the size of the laser spot, respectively. In the Raman setup, the laser diameter was 1 μm, so the laserS equaled 0.785 μm2 and the 4-aminothiophenol drop area on the silicon wafer is approximately 1.2-time larger than that on SERS substrate. Thus, the ratio of RamanN/SERSN was equaled to 8.3 × 105. Therefore, the SERS EF value of Au-Ag-M.w.-20 substrate at the 1367 cm−1 band was ~3.30 × 106.Enhancement factor (EF) was calculated to further quantify the enhancement distribution of the Au-Ag-M.w.-20 substrate according to the following equation :(2)EF=[Ifollowed :(3)N=. These results adequately revealed that the bio-inspired Au-Ag-M.w.-20 substrate possesses high practicality as well as credibility in real detection.In addition, a 6 × 6 µm−10 mg/mL. Therefore, the naturally inspired substrate showed high potential in real-life detection, which was expected to find application in food safety and environmental monitoring.In summary, we have successfully fabricated a kind of ultra-sensitive and flexible SERS platform based on the irregular mantis wing nanoarray by magnetron sputtering technique and physical deposition method. With the 4-aminothiophenol as probe molecules, the Au-Ag-M.w.-20 SERS-active substrate exhibited the most remarkable SERS performance compared with single Cu or Ag nanofilms based hybrids, which could be ascribed to the large-scale “hot spots” that existed between the appropriate nanogaps. In addition, the Au-Ag-M.w.-20 substrate could achieve sensitive SERS measurements with a linear range of the detection of 4-aminothiophenol and the signal reproducibility for R6G was well within 5.53%–7.15% variations. A practical application for the detection of cypermethrin on apple peels was conducted with a LOD reached to be 10


Trójniak” type meads (1:2 honey to water volume) were manufactured from multifloral honey, to which Cornelian cherry fruit juice was added before fermentation. Concentrations of individual iridoids and polyphenols were determined using HPLC analysis with a thermostat refractometric detector, model RID-10A. The total polyphenol content was determined based on testing with Folin–Ciocalteu (F-C) reagent, whereas the antioxidative properties were determined using DPPH• , ABTS•⁺ , and FRAP (ferric reducing antioxidant power) assays. Cultivar of Cornelian cherry fruits influenced both the antioxidative properties and the concentrations of polyphenols and iridoids. The highest concentration of total polyphenols (F-C), accounting for 898.7 mg gallic acid (GAE)/L, was determined in the mead with juice made of red fruits; this mead was also characterized by the strongest antioxidative capabilities measured with ABTS•⁺ and FRAP assays. Among the iridoids determined in the Cornelian cherry meads, loganic acid was found to prevail and its highest concentration, reaching 77.8 mg loganic acid (LA)/L mead, was determined in the mead with the coral-fruit juice. Study results indicate that Cornelian cherry meads have a high content of biologically active iridoids and phenolic acids which display valuable antioxidative properties.In this study, we investigated the effect of Cornelian cherry cultivars differing in fruit color  on physicochemical characteristics, antioxidative properties, and contents of iridoids and polyphenols in meads with the addition of juices made of their fruits. “ An v/v ,6,7; theThe content of biologically active compounds in meads and, hence, their antioxidative capabilities depend on many factors like, e.g., type of honey, heat processing of wort, and parameters of fermentation process, but also the type of additives used, i.e., herbs, spices, or fruits ,9,10. ThFurthermore, these fruits may be used in various branches of the food industry, including production of alcoholic beverages like, e.g., Cornelian cherry liquors ,20,21. W2 emission to be 10% higher than in control samples after 5 days of the fermentation process. The yeast strain did not have a big influence on the dynamics of fermentation of these meads. Roldán et al., 2011, in their research showed bee pollen to be an additive having a strong impact on the improvement of mead fermentation dynamics [Dynamics of the fermentation process is a key parameter enabling the control of its course and determination of its duration. Fermentation of the analyzed meads spanned for 14 days . Introdudynamics .S. bayanus Safspirit Fruit (SF) yeast consumed 20% more of this sugar compared with the S. cerevisiae Safspirit Malt (SM) strain. The aging process also influenced the reduction of the discussed sugars, but to a lesser extent. Evaluation of the carbohydrate profile during fermentation processes allows for controlling their course and determining the effect of technological parameters and substrates used on the extent of extract consumption [The concentrations of fructose and glucose in control samples were 190 and 130 g/L. In worts with the addition of Cornelian cherry juices, the amounts of fructose and glucose were 30 and 20 g/L higher. This is due to the fact that in honey and in Cornelian cherry fruit, which are basic raw materials for our mead production, the main sugars are glucose and fructose ,23. The sumption . Apart fsumption .v/v less than the Cornelian cherry meads manufactured in our experiment [Our study demonstrated ethanol production to be affected by both the type of Cornelian cherry juice and the yeast strain used in mead manufacture. In the presented mead-making technology, alcohol production by the SF yeast was 20 g/L higher compared with the SM yeast, and more ethyl alcohol was produced in the sample with juice from red-fruit Cornelian cherry (MR). Earlier investigations by other authors also have confirmed ethanol concentration in the produced meads to be influenced by the yeast strain and additives used in mead making ,9,22. Hoperiment . Apart from ethanol, which is the major product of alcoholic fermentation, analyses were conducted for concentrations of by-products, like glycerol or acetic acid. After the completed fermentation process, the mean glycerol concentration in the manufactured meads reached 8.9 g/L. After the aging process, it increased by 0.5 g/L on average in all meads. High glycerol production by yeast during fermentation may be caused by ethanolic and osmotic stress. The quantity of produced glycerol may also be affected by the presence of organic acids . Glycerol is of great significance to the quality of meads as it affects their sensory traits, like e.g., fullness of flavor, perception of sweetness, or smoothness ,27. GomeStudy results demonstrate that the meads with the addition of juice from Cornelian cherry fruit had a significantly higher concentration of total polyphenols and significantly stronger antioxidative properties compared with the control meads (WM) without juice addition . The hig• assay were assayed in the meads with the addition of juices from red and coral fruits , whereas a slightly lower activity measured with this method was found in the meads containing juice from yellow fruits (MY). This dependency was confirmed in the analysis of the antioxidative activity of meads conducted with the ABTS•+ assay. In turn, the FRAP assay showed no significant differences in antioxidative activity among the meads with the addition of juices from red fruits and also coral fruit fermented with yeast SM. The highest antioxidative activity was determined in the sample with the addition of juice from red fruits (MR) and fermented with SM yeast; it accounted for 6.2 mmol TE/mL (DPPH• assay), for 7.1 mmol TE/L when analyzed with the ABTS•⁺ assay, and for 8.2 mmol TE/L when measured with the FRAP method and was 39-fold, 21-fold, and 8-fold higher, respectively, than in the mead without fruit juice addition (MW). A slightly lower value of the antioxidative activity was obtained in the meads with juices from coral and yellow fruits; however, it was still significantly higher than in the control variant when measured using all discussed methods. The antioxidative activity and chemical composition of meads is determined by the chemical composition of the raw material and the technology of its processing, by additives used in mead making including fruits or herbs [• and ABTS•+ assays in “trójniak” type meads [•+ assay) [The strongest antioxidative properties measured with the DPPHor herbs , as wellor herbs . The litpe meads . The higp-coumaric acid (p-CuA) and chlorogenic acid (CQA), and the latter was detected only in the wort before beginning the fermentation process. A different group of compounds which affects both the biological properties and quality of plant-based foods is represented by phenolic compounds. Q-3-Glucuronoide (Q-3-glcr) was the only one among the quantified flavonols which was detected in all meads with the addition of Cornelian cherry fruit juices , phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), and flavonoids  . After at juices . In turn•), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), dimethyl sulfoxide (DMSO), FeCl3, acetonitrile, formic acid, sulfuric acid, sodium hydroxide, and hydroxymethylfurfural (HMF) were acquired from Sigma-Aldrich . Acetic acid was obtained from Chempur . Acetonitrile for HPLC was purchased from POCh . Loganic acid (LA), loganin (Lo), Sweroside (S), gallic acid (GA), ellagic acid (EA), 5-O-caffeoylquinic acid , p-coumaric acid (p-CuA), quercetin 3-O-glucoside (Q glc), kaempferol 3-O-glucoside (Kf glc), cyanidin 3-O-glucoside (Cy glc) were purchased from Extrasynthese . All reagents were of analytical grade.1,1-Diphenyl-2-picrylhydrazyl radical (DPPHSaccharomyces bayanus Safspirit fruit (SF) and Saccharomyces cerevisiae Safspirit malt (SM) yeast from the Fermentis company . Before being inoculated, dried yeast was rehydrated in distilled water at a temperature of 25 °C for 30 min.The biological material were The studies used the same plant material as that described in detail in the publications by Kawa-Rygielska et al. .Rapeseed honey was stirred with water in the ratio of 1:2 (honey/water). The resultant wort was boiled at a temperature of 100 °C for 1.5 h, with regular stirring and skimming off the scum. Afterwards, the mixture was cooled and for all mead variants, the initial extract content in the wort was set at 34° Bx. Wort was divided into four variants : control2 emission in time. To this end, the fermentation samples were weighed on a WTB 2000 scale by RADWAG company  every 24 h throughout the alcoholic fermentation. Extract content was determined with a Densito 30 PX densitometer by Mettler-Toledo company . The samples were earlier centrifuged using an MPW-351R laboratory centrifuge , 6000× g, 10 min) by MPW MED. INSTRUMENTS company . Extract content was measured in the resultant supernatant at a temperature of 20 °C. The pH value was measured with an MP 220 pH-meter by Mettler Toledo company .Dynamics of the fermentation process were controlled based on CO2SO4; and thermostat refractometric detector at 50 °C. Concentrations of compounds were determined based on a five-point calibration curve integrated in Chromax 10.0 software by Pol-Lab .The concentrations of sugars , alcohol, organic acid, and glycerol were determined by means of HPLC . CentrifThe total polyphenolic content of the meads was determined using Folin–Ciocalteu’s phenol reagent (F-C) . Detaile• and ABTS assay [The antiradical and antioxidant activity was determined using DPPHTS assay ,44,45 anTS assay . All tesTS assay . The datO-caffeoylquinic acid, p-coumaric acid and p-coumaric acid derivatives as p-coumaric acid, quercetin 3-O-glucuronide as quercetin 3-O-glucoside, aromadendrin 7-O-glucoside and kaempferol 3-O-galactoside as kaempferol 3-O-glucoside, and anthocyanins as cyanidin 3-O-glucoside. The results are expressed as mg per liter of the mead.The analysis was previously described by Kucharska et al. . The higp < 0.05).Mean deviations are shown in tables. Selected data were processed using Statistica 13.5 software  for calculating a one-factor analysis of variance (ANOVA) with significance level α = 0.05. Differences between means were tested using the Duncan test (The study showed the possibility of using the analyzed cultivars of Cornelian cherry for the manufacture of fruit meads with health benefits. Red juice of the “Podolski” variety greatly enriched mead with compounds with strong antioxidant activity. The addition of Cornelian cherry allowed us to obtain a new alcoholic drink rich in iridoids. The content of active compounds in meads depends on the variety of Cornelian cherry fruits that is added to their production. The red fruits significantly increase the content of these compounds in the final product. Cornelian cherry mead as a fermented beverage can be a good alternative to red grape wines, if it is consumed regularly but with restraint.


The two fragments NGFP and CGFP do not assemble a fluorescent GFP protein when produced in trans, but they assemble a fluorescent GFP when fused to interacting proteins. To adapt the method to high salt, we used the genes encoding two fragments of the salt-stable mGFP2 to construct four vector plasmids that allow an N- or C-terminal fusion to the two proteins of interest. To avoid a hindrance in the assembly of mGFP2, the fusion included a linker of 15 or 19 amino acids. The small gas vesicle accessory protein GvpM and its interaction partners GvpH, GvpJ, and GvpL were investigated by split-GFP. Eight different combinations were studied in each case, and fluorescent transformants indicative of an interaction were observed. We also determined that GvpF interacts with GvpM and uncovered the location of the interaction site of each of these proteins in GvpM. GvpL mainly interacted with the N-terminal 25-amino acid fragment of GvpM, whereas the other three proteins bound predominately to the C-terminal portion. Overall, the split-GFP method is suitable to investigate the interaction of two proteins in haloarchaeal cells. In future experiments, we will study the interactions of the remaining Gvps and determine whether some or all of these accessory Gvp proteins form (a) protein complex(es) during early stages of the assembly of the gas vesicle wall.Several extremely halophilic archaea produce proteinaceous gas vesicles consisting of a gas-permeable protein wall constituted mainly by the gas vesicle proteins GvpA and GvpC. Eight additional accessory Gvp are involved in gas vesicle formation and might assist the assembly of this structure. Investigating interactions of halophilic proteins  Halobacterium salinarum. Gas vesicles enable the cells to float to the surface of the brine where light and oxygen concentrations are optimal for growth. The gas vesicle wall consists exclusively of aggregated proteins, and we are interested in investigating the protein–protein interactions required during their formation. Hbt. salinarum lives at salt concentrations of up to 5.3 M NaCl and uses the salt-in strategy to adapt to its salty environment. Isoosmotic potassium chloride concentrations are present in the cytoplasm, and the structure and function of most haloarchaeal proteins thus depends on salt. The gas vesicles are easy to isolate by lysis of the cells in water followed by centrifugation-enhanced flotation; they are stable in water or detergent solutions and only dissolve in 80% formic acid  and challenged in vivo by analyzing the effect of single amino acid (aa) substitutions on gas vesicle formation in Hfx. volcanii ΔA+Amut transformants   . Some muructures . The halclusters .gvp) genes arranged in two oppositely oriented transcription units, gvpACNO and gvpDEFGHIJKLM . In the case of GvpA, the β-strands most likely constitute the hydrophobic interior surface of the gas vesicle wall and prevent the precipitation of water molecules in the interior. The functions of GvpJ and GvpM, and of the other accessory Gvp are not yet known. They could be minor constituents of the gas vesicle wall, or act as scaffolding protein or chaperone to keep the hydrophobic GvpA in solution before incorporated in the wall. A crucial step in the formation of gas vesicles is the start of the Gvp aggregation. Since gvpFGHIJKLM is transcribed early in growth, the proteins encoded might be required in early stages of gas-vesicle assembly. All of these accessory Gvp are small, ranging in size from 9.2 (GvpM) to 32 kDa (GvpL). The involvement of GvpM in an early stage of gas vesicle formation was also proposed since point mutations in GvpM studied in ΔM+Mmut transformants resulted either in gas vesiculated (Vac+) or Vac negative cells , and isolation under denaturing conditions in 8 M urea using a Ni-NTA matrix . In addition, fragments of GvpM were used to define the interaction sites of these four Gvp proteins with GvpM in further detail.To investigate protein–protein interactions in nd yeast . The gresmRS-GFP  with an Escherichia coli strains One Shot Top10 (Invitrogen by Life Technologies) and GM1674 (−dam)  tryptone, 0.3% (w/v) yeast extract, and 0.02% (w/v) histidine. For solid medium, 1.8% (w/v) agar was added. To select Hfx. volcanii transformants, 0.2 μg/ml novobiocin (for selection of pJAS35) and 6 μg/ml mevinolin (for selection of pWLfdx) were supplemented. Incubation was done at 42°C, and with the split-GFP expressing cells at 37°C or 37°C followed by 30°C as described in the “Results” section. Cultures on solid medium were incubated in humid atmosphere for 3–7 days, whereas liquid cultures were incubated 1–4 days with shaking at 180 rpm.The 4 (dam−)  were grongfp- or cgfp-portion of mgfp2 at the 5′- or 3′-terminus of the respective gvp reading frame under investigation. The plasmids pJAS-NGFP-Nterm and pJAS-NGFP-Cterm are based on the shuttle vector pJAS35 . Both vectors occur in similar copy numbers per cell. Nterm describes the fusion of the respective gfp fragment at the 5′-terminus of gvp and Cterm the fusion at the 3′-end. GFP was split between amino acid residues 157 and 158, leading to NGFP (17.7 kDa) and CGFP (9.0 kDa). The synthetic oligonucleotides used to amplify the respective fragments (Supplementary Table S1) included also a linker sequence. The linker region in pJAS-derived vectors [(GGSGSGS)2] is 14 aa in size, and the linker region in the pWL vectors 16 aa [(GGSG)4]. Specific restriction sites were added leading to linker lengths of 15 to 19 aa . The plasmid carrying mgfp2 served as template for amplification of both fragments. The BspHI-ngfp-link-BlpI and the NcoI-link-ngfp-KpnI fragments were inserted in the expression vector pJAS35, and the NcoI-link-cgfp-KpnI and the NcoI-cgfp-link-KpnI fragments in pWLfdx . In each case, the expression of the reading frames is driven by the strong fdxP promotor . The NcoI-gvp-BlpI fragment was inserted in pJAS-NGFP-Nterm and pJAS-NGFP-Cterm . For the insertion of gvp in pWL-CGFP-Cterm, the restriction sites NcoI and BamHI were used, and BamHI and KpnI for the insertion in pWL-CGFP-Nterm. The correct fusion of each gvp to ngfp or cgfp was confirmed by DNA sequence determination. To overcome a restriction barrier in Hfx. volcanii, the plasmids were passed through the E. coli GM1674 (−dam) to obtain demethylated DNA. Hfx. volcanii was transformed simultaneously with the two vector plasmids as described previously  and re-suspended in 2–3 ml lysis buffer  glycerol, 50 mM Tris-HCl pH 8.0). Cell lysis was achieved by sonication on ice . The lysate was cleared by centrifugation  and dialyzed against 10 mM Tris-HCl pH 7.2 for 2 h to eliminate salts. After dialysis, 20 μg of protein were separated by SDS-PAGE  using the PerfectBlueTM ‘Semi-Dry’-Blotter. The membrane was dried for 1 h, reactivated with 100% methanol, washed for 2 min in PBS  and blocked for 1 h with Odyssey Blocking Buffer (LI-COR). The membrane was incubated overnight with the respective Gvp antiserum raised against GvpH, F, J, L, or M (® 20. Incubation with the secondary antibody IRDye 800CW (LI-COR) was done for 1–2 h and the membrane was washed four times for 5 min with PBS + 0.1% (v/v) Tween® 20. To remove excessive Tween® 20, the membrane was washed with PBS. The secondary antibody is coupled with a fluorophore detectable in the near-infrared range of 800 nm with an Odyssey Fc Imager (LI-COR).The presence of N/CGFP-Gvp fusion proteins was confirmed by Western analysis. Total protein was isolated from 50 ml cultures in the exponential growth phase. The cells were harvested by centrifugation , washed with 1 ml basal salts , and re-suspended in 500 μl basal salts. Samples of 300 μl brought to OD600nm 1 were analyzed in a 96-well plate and evaluated using the Fujifilm science lab image gauge ver. 4.24 software. Fluorescence measurements are given in light absorbing units (LAU) per mm2 (Supplementary Table S2). All experiments were performed with two biological samples and three technical replicates. The relative fluorescence (rf) was calculated using the formula given below and the standard deviation and the p-values were calculated using Student t-testTo demonstrate the protein–protein interaction 600nm 1.5 and investigated. A Leica TCS SP5 II confocal microscope in combination with Leica application suite software was used for analysis. Image processing was done by the software Fiji.To investigate the cell fluorescence a Confocal Laser Scanning Microscopy (CLSM) was used. The transformants were grown to ODin vivo using a modified salt-adapted split-GFP.In this study we investigated the protein–protein interactions of several accessory Gvp proteins involved in gas vesicle formation fdx (CGFP), initially providing a 7-aa linker between Gvp and N-/CGFP. Both plasmids occur in similar copy numbers per cell, and the expression of the inserted reading frames is driven by the ferredoxin promoter in both cases . The resulting four plasmids allow the fusion of the reading frame of interest to ngfp or cgfp at the 3′- or the 5′-terminus. Four combinations of the “empty” vectors containing ngfp or cgfp but lacking a gvp reading frame were tested for an assembly of mGFP2 . None of these transformants indicated a higher fluorescence than Hfx. volcanii demonstrating that the self-assembly of mGFP2 did not occur (Supplementary Table S2).The modified green fluorescent protein mGFP2  was split between β-strands 7 and 8 to obtain the N-terminal fragment NGFP (residues 1–157) and the C-terminal CGFP (residues 158–239). The reading frames encoding these fragments were inserted in the compatible expression vectors pJAS35 (NGFP) and pWLth cases . The reagvpM or gvpL fused to the n/cgfp fragments were tested in Hfx. volcanii. The resulting fusion proteins NGFPM, CGFPM, MNGFP, MCGFP, NGFPL, CGFPL, LNGFP, or LCGFP carried NGFP or CGFP at the N- or C-terminus of GvpM or GvpL, and will be further described as NM, CM, MN, MC, NL, CL, LN, LC for convenience. The fluorescence was initially measured in cells grown at 37°C, but the fluorescence emitted was relatively low . To enhance the protein folding at lower temperatures, the cultures were grown to OD 1 at 37°C for 1 day to obtain sufficient cell mass, followed by incubation of the culture at 30°C overnight. This procedure increased the fluorescence signal threefold  and demonstrated that the slower growth at 30°C helps folding and assembly of split-GFP. All eight L/M combinations were tested under the latter condition, and three of them yielded fluorescent transformants, i.e., NL/MC , LN/MC (rf 1.6), and LC/MN (rf 1.3) . Inspecting the transformants by fluorescence microscopy determined that the entire cells of NL/MC were fluorescent, whereas single fluorescent foci were observed with LC/MN and LN/MC transformants , presumably causing the large difference in rf. The transformants were also investigated by Western analysis using an antiserum detecting GvpM or GvpL to ensure that the fusion proteins were produced . The NM, MN and MC proteins were well detectable, whereas CM was not found . It is likely that the lack of fluorescence of LN/CM and NL/CM transformants was due to the undetectable amount of CM. In the case of GvpL, any of the NGFP-GvpL fusion proteins were observed, whereas the various CGFP-GvpL fusions were more difficult to detect since unspecific reactions of the GvpL antiserum occurred in the expected size range of 40–45 kDa . Overall, an assembly of mGFP2 occurred mainly when N/CGFP was fused to the C-terminus of GvpM, whereas the N-terminal fusions of N- or CGFP yielded a low or undetectable fluorescence . The latter results suggested that the N-terminal fusion might hinder the assembly of mGFP2, and that the N-terminal region of GvpM might be required for the GvpL interaction.Eight combinations of the plasmids carrying Figure 4). The two N-terminal deletion variants MΔ5N and MΔ10N, as well as the C-terminal deletion variant MΔ10C have been already tested for gas vesicle formation in ΔM+Mmut transformants . In contrast, a 10-aa deletion at the C-terminus results in gas-vesicle containing Vac+ ΔM+MΔ10C transformants. Further C-terminal deletions (up to 30 aa) were constructed and investigated for gas vesicle formation . Western analysis demonstrated that all GvpM deletion variants were stable and detectable . Transmission electron microscopy showed that the ΔM+MΔ10C and ΔM+MΔ20C transformants contained many gas vesicles, whereas a few cells of the ΔM+MΔ25C transformants contained a single gas vesicle only, and ΔM+MΔ27C or ΔM+MΔ30C transformants were Vac negative . These results implied that a large portion of the C-terminus of GvpM including helix α3 (aa 65–84) is not required for gas vesicle formation, whereas the N-terminal region is important.To determine the importance of the terminal regions of GvpM for gas vesicle formation and for the interaction with GvpL, different GvpM deletion variants were investigated  yielded 72–76% of the fluorescence obtained with NL/MC transformants, and only the fluorescence of the NL/MΔ30CC transformants was reduced to 57% . These results implied that deletions at the C-terminus of GvpM had only a minor effect on the interaction with GvpL. The reduction to 57% with MΔ30C could be due to the relatively large deletion encompassing helix α3 and the loop between α2 and α3; this might affect the GvpM structure and also the L–M interaction. In the case of the N-terminal deletions, the fluorescence of NL/MΔ5NC transformants was reduced to 46%, and in NL/MΔ10NC transformants even to 15%, demonstrating a strong effect on the interaction with GvpL .The various GvpM deletion variants were used to test the interaction with GvpL in the combination Figure 4A). Each of these fragments was fused to NGFP or CGFP at the N- or C-terminus and tested with the respective N/CGFP-GvpL fusions in Hfx. volcanii . In the case of the N-terminal fragment M(25N), four of the eight combinations yielded highly fluorescent cells (rf 21–51), strongly supporting the idea that this fragment mediates the interaction with GvpL. In contrast, the eight combinations of the central fragment M(25–59) tested by split-GFP yielded no detectable fluorescence, and also the combinations including the C-terminal portion M(25C) showed no fluorescence except for LC/NM(25C) (rf 16) . These results underlined that GvpL contacts GvpM preferentially in the N-terminal 25-aa. Compared to the interaction study using the full-length GvpM protein (rf 12), the fluorescence was much higher with M(25N) (rf 51), demonstrating that a smaller fragment is very useful to determine an interaction site.To challenge the hypothesis that the interaction of GvpL occurs in the N-terminal portion of GvpM, three fragments of GvpM were investigated, i.e., M(25N) encompassing the N-terminal 25 aa including helix α1, M(25–59) containing the central portion including the β-sheets plus α2, and the C-terminal fragment M(25C) with helix α3 . A low fluorescence (up to rf 1.5) occurred when fragment M(25N) was used, and no fluorescence was detectable in all combinations of the central portion M(25–59). However, the C-terminal M(25C) fragment yielded a high fluorescence in HC/NM transformants , suggesting an interaction of GvpH with the C-terminal portion of GvpM. In the case of the GvpM-related GvpJ (12 kDa), a very low fluorescence was observed with the NJ/MC transformants carrying the full-length GvpM . A similarly low fluorescence was observed in all cases when the N-terminal fragment M(25N), or the central fragment M(25–59) were tested with GvpJ . The C-terminal fragment M(25C) yielded a slightly enhanced fluorescence (rf 1.9) with JC/NM(25C) transformants. Analyses using His-tagged GvpJ demonstrated the J–M interaction, and the results presented here suggested that GvpJ might contact the C-terminal portion of GvpM .The 19.8-kDa GvpH is able to prevent the aggregation of GvpM in haperone . AnalyseNF/MC transformants, and also with some combinations of M(25N) . No fluorescence was detectable with the central region M(25–59), but a high fluorescence (rf 12) was obtained with FC/NM(25C) transformants . The latter result implied that GvpF interacts with the C-terminal portion of GvpM. To support these results, the N- and C-terminal deletion variants of GvpM were tested with the split-GFP method. Using MΔ5N for the investigation of the F–M interaction, the fluorescence was very similar to GvpM wild type . The NF/MΔ10NC transformants yielded a strongly reduced fluorescence (33% of the GvpM wild type) implying that the sequences deleted are involved in the interaction with GvpF. All transformants harboring a C-terminal deletion in GvpM (MΔ10C through MΔ27C) showed reductions to 63 and 53% of the wild type, and the fluorescence of the NF/MΔ30C transformants was reduced to 31% . Overall, these results supported the hypothesis that the interaction F–M mainly occurs in the C-terminal portion of GvpM.Investigating the 23.7-kDa GvpF for interaction with GvpM yielded a low fluorescence (rf 1.6) in C/-, HC/-, or JC/NM(25C), i.e., when NGFP was fused to the N-terminus of M(25C) and CGFP to the C-terminus of the accessory protein tested.In summary, our data implied that the accessory proteins GvpF, GvpH, and GvpJ interact predominantly with the C-terminal portion of GvpM. In each case, the highest fluorescence was achieved in the combination Ffdx  protein–protein interactions are important to understand the protein aggregations that occur during the formation of gas vesicles in haloarchaea. The split-GFP method has been applied in bacteria and yeast to analyze the interactions of proteins, e.g., involved in cell division . Since Gfdx . In bothFigure 1), whereas GvpL is with 32 kDa relatively large . Eight combinations of the four different N/CGFP fusion variants were tested in Hfx. volcanii. The highest fluorescence was obtained in the combination NL/MC , and to a less extent with LN/MC, and LC/MN, whereas all other combinations did not result in a detectable GFP fluorescence. Thus, it is important to analyze the different combinations of N/CGFP fusions, since GFP assembly of the two fragments depends on physical constraints of the interacting proteins. The highly fluorescent NL/MC transformants contained the assembled mGFP2 distributed in the cells, whereas other transformants harbored aggregated mGFP2 as a single fluorescent focus per cell, presumably caused by an aggregation of GvpM. The N- or C-terminal fragment of GvpM lacking the hydrophobic central portion always yielded fully fluorescent cells when tested by split-GFP (data not shown). Since mainly C-terminal fusions of the N/CGFP-fragments to GvpM yielded fluorescent cells, we hypothesized that the N-terminus of GvpM was involved in the L–M interaction. Testing the N-terminal 25-aa of GvpM confirmed that the contact site is located here. In addition, another less distinct interaction site might be located in the C-terminal portion of GvpM. The smaller fragments were excellent interaction partners for GvpL, since the structural constraints for the assembly of mGFP2 are lower. This was already shown by testing small leucine-zipper regions of a transcriptional regulator in E. coli for an interaction  and HC/NM(25C) and to a less extent in JC/NM(25C), whereas the central portion of GvpM yielded no fluorescent transformants. It is interesting to note that GvpF (23.9 kDa) and GvpL (32 kDa) have very similar 3D-structures. A crystal structure of GvpF derived from the cyanobacterium Microcystis aeruginosa is available and shows two structurally distinct domains displaying an α+β structure . Both 3D-models are similar and contain two domains with α+β folds . Nevertheless, the similarity of their amino acid sequences is not very high (35%) . Our analysis on the F–M and L–M interactions implied that both proteins bind to different portions of GvpM, since GvpL showed a high affinity to the N-terminal portion, and GvpF to the C-terminal portion of GvpM. It is possible that GvpL and GvpF bind simultaneously to GvpM, and a possible complex formation should be investigated. The interaction of GvpF with the C-terminal portion of GvpM is somewhat surprising since this region is not required for gas vesicle formation; most of this region could be deleted without the loss of gas vesicle formation. A sequence alignment of GvpM derived from the p-vac region (pGvpM was investigated here) and cGvpM encoded by the second gas vesicle region c-vac of Hbt. salinarum shows that the last 11 aa of pGvpM are not found in cGvpM . This observation suggests that the binding site(s) of the three accessory Gvp proteins might be located further upstream, possibly in the loop between the helices α2 and α3. Also here, point mutations should be tested to determine the interaction sites more precisely.The C-terminal fragment of GvpM comprising the helix α3 appeared to interact with the accessory proteins GvpF and GvpH, and presumably with GvpJ. A high fluorescence was observed in the combinations Ftructure . This crGFP+H transformants in comparison to MGFP transformants using a fluorescent GvpM–GFP fusion . The analysis of GvpJ by split-GFP detected a low fluorescence in transformants harboring the full-length GvpM or fragment M(25N). Only transformant JC/NM(25C) yielded a nearly fourfold higher fluorescence than transformants harboring the full-length GvpM. However, the relative fluorescence was much lower compared to the fluorescence obtained for the F–M and H–M interaction . The low fluorescence observed with the J–M interaction is in contrast to the strong selection of GvpJ from a Hfx. volcanii lysate by GvpMHis bound to Ni-NTA . Also, it is possible that the interaction requires not a consecutive aa sequence, but amino acids that occur in close location at the surface. In addition, the low fluorescence could be due to the hydrophobic nature of GvpJ. The aggregation could influence the availability of GvpJ for the interaction with GvpM.GvpJ (12 kDa) is a small, hydrophobic protein related to GvpA and GvpM and structural modeling suggests a similar 3D-structure  a larger protein complex. The split-GFP method will be applied to determine additional interactions between the accessory Gvp and also with GvpA. In addition, we will investigate whether the accessory Gvp proteins form a larger protein complex during gas-vesicle assembly, but this requires different methods.In summary, we demonstrated that the interaction of haloarchaeal proteins can be studied by split-GFP KW and FP planned the study, discussed the results, and wrote the manuscript. KW performed the analysis. JB designed mGFP2 used for the construction of split-GFP. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Inflammation is closely implicated in the process of osteoarthritis (OA) and affects disease progression and pain. Herein, the present study explored the effect of microRNA-26a (miR-26a) on the synovial inflammation and cartilage injury in OA, with the involvement with the NF-κB signaling pathway.Methods: Rat models of OA were established by anterior cruciate ligament transection, which were then treated with miR-26a mimics/inhibitors or BMS-345541 (inhibitor of NF-κB pathway). The expression of miR-26a and activator proteins of NF-κB pathway (P-IκBα and P-P65) in synovial tissues was determined. Hematoxylin-eosin (HE) staining was adopted to observe pathological changes of knee joints, synovial tissues, and cartilage of femoral condyle. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the apoptosis of synoviocytes and chondrocytes.Results: Poorly expressed miR-26a and increased protein levels of P-IκBα and P-P65 were identified in synovial tissues of OA rats. Besides, OA rats showed obvious synovial tissue hyperplasia, inflammation and cartilage injury of femoral condyle, as well as increased inflammation and cartilage injury scores, and apoptosis of synoviocytes and chondrocytes. In response to miR-26a mimics, protein levels of P-IκBα and P-P65 were reduced; meanwhile, synovial tissue hyperplasia, inflammation and cartilage injury of femoral condyle were ameliorated, with decreased inflammation and cartilage injury scores, and apoptosis of synoviocytes and chondrocytes.Conclusion: MiR-26a suppressed the activation of the NF-κB signaling pathway, by which mechanism the synovial inflammation and cartilage injury in OA rats were alleviated. Osteoarthritis (OA) is the most common type of arthritis worldwide, and it is a major contributor to pain and disability among the elderly population . OA mainMiRNAs are a class of small, non-protein-coding RNAs functioning as gene regulators, which control target genes and diverse biological pathway , 11. MicTotally, 72 clean and healthy Sprague–Dawley (SD) rats, weighing 200 ± 20 g, including 24 male rats and 24 female rats, were purchased from the Experimental Animal Center (License number: SCXK[Chongqing]2007-0005) of the Third Military Medical University . The rats were allowed to acclimatize to environment and housed in a clean animal room, with temperature at 18–24°C, 12 h light/dark cycle and free access to food and water. All experimental protocols used in the present study were approved by the Ethics Committee for Animal Experimentation of the First Affiliated Hospital of Bengbu Medical College.n=8), OA group (OA rats without any treatment), NC group (OA rats injected with miR-26a NC sequence through knee joint cavity), BMS-345541 group , and miR-26a inhibitors + BMS-345541 group . MiR-26a NC sequence, miR-26a mimics, and miR-26a inhibitors were purchased from Shanghai GenePharma Co., Ltd. . BMS-345541 was purchased from Sigma .In order to observe the role of miR-26a in synovitis and cartilage damage of OA rats, we allocated these rats into five groups with eight rats in each group: normal group , OA group (OA rats without any treatment), negative control (NC) group (OA rats injected with miR-26a NC sequence through knee joint cavity), miR-26a mimics group (OA rats injected with miR-26a mimics through knee joint cavity), and miR-26a inhibitors group (OA rats injected with miR-26a inhibitors through knee joint cavity). In order to further observe whether miR-26a has effect on synovitis and cartilage damage of OA rats by regulating the NF-B signaling pathway, we allocated the remaining rats into four group (Anterior cruciate ligament (ACL) transection was employed to establish rat models of OA. Except for those in the normal group, the rats were fixed on the operation table in a supine position. A 1 cm incision was made in the medial side of the right knee joint to expose the medial collateral ligament, which was then cut off. The joint capsule was opened to examine whether there was primary lesion in the joint cavity. Then the ACL was transected and the medial meniscus was completely cut off. The incisions were sutured layer by layer, bandaged under sterile conditions, and fixed. Following the operation, antibiotic  was administered the next day to prevent infection. 1 week after the operation, rats in the OA group and the normal group were conventionally kept. The other groups were treated separately with the same housing conditions.On the 28th day of experiment, five rats from each group were collected and killed by cervical dislocation. The right knee joint specimen were taken and observed by the naked eye, the muscles around the knee joint was quickly removed to remove the knee joint. The femoral condylar cartilage was removed by circular incision along the femoral edge of the articular capsule. In addition, the femur and tibial fibula were cut off along the bone surface, the joint capsule was cut longitudinally, and the synovial tissue was separated. Then the cartilage and synovial tissue specimens were stored in liquid nitrogen, which were used for pathological detection and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining experiment. The knee joints of remaining SD rats were quickly put into liquid nitrogen for later detection of the expression of miR-26a, P-I κBα, and P-P65 in cartilage and synovial tissues.The specimens of the joints were fixed with a sharp knife to form a thick slice of 3–5 mm, fixed in 10% formaldehyde solution, and then put into the ethylene diamine tetraacetic acid (EDTA) for routine decalcification. According to the procedure of an automatic dehydrator, the specimens were dehydrated with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, n-butanol, anhydrous ethanol I and anhydrous ethanol II, each for 2 h, cleared with xylene II for 2 h, embedded with paraffin I and paraffin II, each for 2 h (60–62°C). After conventional paraffin embedding, the sharp blade was used to keep a 4 μm thick tissue slice, and the tissue slice was automatically dried in the whole machine, and then stored at room temperature, to be used.No less than three slices of synovial and cartilage tissues were taken from each rat in each group. Paraffin sections were dewaxed and hydrated. The pathological changes of synovial and cartilage tissues of each group were analyzed by hematoxylin-eosin (HE) staining under a light microscope. The HE staining of femoral condylar cartilage was observed and scored by two technical experimenters who were not familiar with the study under a microscope. Three visual fields were selected for each picture. Mankin scores were assessed for articular cartilage by the assigned experimenter. The 0–2 scores represent normal cartilage; 3–5 scores represent fibrosis on the surface of cartilage; 6–7 scores represent medium cartilage injury; 8–10 scores represent severe cartilage injury; above 10 scores represent complete loss of cartilage . Three fThe paraffin-embedded sections of articular synovial tissues and cartilage tissues of knee joints were collected to perform TUNEL staining. Along the synovial tissue band and cartilage tissue band, the number of apoptotic cells was counted (×200). A total of ten fields were counted, followed by recording and calculating the mean number of apoptotic cells in each field. The apoptotic synoviocytes and chondrocytes were brown yellow in color. Apoptotic index (AI) =  × 100%.t) value of each reaction tube was recorded. The data were analyzed by 2−ΔΔCt, which represents the multiple proportion of gene expression of the experimental group to the control group. The formula was ΔΔCt = [ΔCttarget gene − ΔCtreference gene]experimental group − [ΔCttarget gene − ΔCtreference gene]control group. The experiment was conducted in triplicate with mean value calculated.On the 28th day of experiment, Trizol method  was adopted to extract total RNA of half of synovial tissues of knee joints from the remaining three rats of each group. The ultraviolet analysis and formaldehyde gel electrophoresis were employed to confirm the high quality of RNA. Then 1 μg RNA was collected and avian myeloblastosis virus (AMV) reverse transcriptases were used for reverse transcription to obtain cDNA. The primers of PCR were designed and synthesized by Invitrogen  , with glThe protein of half of synovial tissues of knee joints from the remaining three rats of each group was extracted. The protein concentration was determined according to the bicinchoninic acid (BCA) protein assay kit . The extracted protein added with uploading buffer was boiled at 95°C for 10 min, with 30 μg for each well. Subsequently, 10% PAGE  was employed to separate proteins, with electrophoresis voltage 80 V transferring to 120 V by wet transfer method. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes with transferring voltage of 100 mV for 45–70 min. The membranes were blocked with 5% BSA for 1 h. Primary antibodies of P-IκBα , P-P65 , and β-actin  were added and incubated at 4°C overnight, followed by washing three times (5 min per wash) with Tris-buffered saline with Tween 20 (TBST). Corresponding secondary antibodies  were added and incubated for 1 h. The membranes were washed for three times with 5 min for each time. Chemiluminescence reagents were employed to develop images. β-actin was considered as an internal reference. The images of the gels were captured in a Bio-Rad Gel Doc EZ Imager . The gray values of target protein bands were analyzed by ImageJ software . The experiment was conducted in triplicate with mean value calculated.t test. Comparison among multiple groups was analyzed by one-way analysis of variance. Pairwise comparison was conducted by the least significant difference (LSD) t test. Two-sided P-values <0.05 were considered to indicate statistically significant differences.SPSS 21.0  was employed to analyze data in the present study. Measurement data are expressed by mean ± standard deviation. Normally distributed measurement data between two groups were analyzed by the P<0.05), suggesting that miR-26a expression is associated with OA.RT-qPCR was used to examine the expression of miR-26a in the synovial tissues of knee joints of rats A. A loweP>0.05). Compared with the OA group, the miR-26a expression was increased in the miR-26a mimics group and reduced in the miR-26a inhibitors group (both P<0.05), indicating that the interference of miR-26a mimics and inhibitors was successful . When compared with the OA and the NC groups, the AI of synoviocytes of knee joints was decreased in the miR-26a mimics group and increased in the miR-26a inhibitors group . These results indicated that overexpressed miR-26a could suppress the synovial inflammatory injury in the knee joints of rats with OA.TUNEL staining was adopted to observe the apoptosis conditions of synoviocytes in knee joints of rats D,E. ScatP<0.05). In the miR-26a mimics group, the Mankin scores of cartilage of femoral condyle were lower than those of the OA group (P<0.05). The miR-26a inhibitors group had significantly increased Mankin scores of cartilage of femoral condyle as compared with the OA group (P<0.05). These results indicated that overexpressed miR-26a reduced cartilage injuries of knee joints of rats with OA.The pathological changes of femoral condyle cartilage of femoral condyle were observed A. In theP<0.05). When compared with the OA and NC groups, the AI of chondrocytes of knee joints was decreased in the miR-26a mimics group and increased in the miR-26a inhibitors group . These results indicated that overexpressed miR-26a could suppress the pathological injury in the cartilage of knee joints of rats with OA.The apoptosis of femoral condyle chondrocytes in knee joints of rats were evaluated by TUNEL staining C,D. CompP<0.05), suggesting that the NF-κB signaling pathway was aberrantly activated in OA models. No significant differences were identified in protein levels of P-IκBα and P-P65 in synovial tissues of knee joints between the OA group and the NC group (both P>0.05). The protein levels of P-IκBα and P-P65 in synovial tissues of knee joints was reduced in the miR-26a mimics group and increased in the miR-26a inhibitors group, as compared with the OA group , indicating that miR-26a could inhibit the aberrant activation of NF-κB signaling pathway in OA rats.In order to analyze the status of NF-κB signaling pathway in OA and the control of miR-26a on NF-κB signaling pathway, we adopted Western blot assay to examine the protein levels of activating factors of NF-κB signaling pathway (P-IκBα and P-P65) in synovial tissues of knee joints A,B. The BMS-34554 was employed to inhibit the activation of NF-κB signaling pathway in OA rats, under which condition, the protein levels of P-IκBα and P-P65 in synovial tissues of knee joints was reduced. In response to the combined treatment of miR-26a inhibitors and BMS-34554, protein levels of P-IκBα and P-P65 in synovial tissues of knee joints was higher than those in the BMS-34554 group, suggesting that miR-26a regulated the status of NF-κB signaling pathway C,D.The NF-κB signaling pathway was aberrantly activated in OA rats. When BMS-34554 was employed to inhibit the activation of NF-κB signaling pathway in OA rats, the synovial hyperplasia, articular cartilage injury and wear were alleviated. The combined treatment of miR-26a inhibitors and BMS-34554 increased the synovial hyperplasia, articular cartilage injury and wear, as compared with the treatment of BMS-34554; while no differences were found as compared with the OA and the NC groups A.P<0.05).The pathological changes of synovial tissues of knee joints were observed B,C. CompP<0.05). Compared with the OA and NC groups, the AI of synoviocytes was not different in the miR-26a inhibitors + BMS-345541 group (P>0.05). These findings implied that miR-26a could reduce synovial inflammation in knee joint of OA rats via inhibiting NF-κB signaling pathway.TUNEL staining was used to evaluate apoptosis of synoviocytes in knee joints D,E. The P<0.05). In the miR-26a inhibitors + BMS-345541 group, the staining of the femoral condyle cartilage matrix in the knee joints was uneven, with irregularly arranged chondrocytes; the cartilage injury was similar to the OA and NC groups; Mankin scores were increased as compared with the BMS-345541 group (P<0.05)  A,B.P<0.05). Compared with the BMS-345541 group, the miR-26a inhibitors + BMS-345541 group showed increased AI of chondrocytes in knee joints (P<0.05). These findings implied that down-regulation of miR-26a expression could deteriorate the pathological injuries of cartilage in knee joints; whereas, blockade of the NF-κB signaling pathway could reverse the effect of miR-26a inhibitors.Meanwhile, TUNEL staining was used to observe the apoptosis of chondrocytes in knee joints of rats C,D. The OA, common in older adults, is a highly prevalent, chronic, degenerative condition inflicting joint inflammation, pain and even disability on patients . It is rThe OA models of knee joints were established in rats by ACL transection. In the synovial tissues of OA rats, obvious synovial tissue hyperplasia, inflammation, and cartilage injury of femoral condyle were observed; meanwhile, we found increased inflammation and cartilage injury scores and apoptosis of synoviocytes and chondrocytes. Benito et al.  stated tCorroborating with our study, another study indicated that the low expression level of miR-26a was associated with increased chronic inflammation in the chondrocytes in obese mouse model; whereas, up-regulated miR-26a expression attenuated activation of NF-κB and secretion of pro-inflammatory cytokines in articular chondrocytes . Based oIn order to analyze the status of NF-κB signaling pathway in OA and the control of miR-26a on NF-κB signaling pathway, we adopted Western blot assay to examine the protein levels of activating factors of NF-κB signaling pathway (P-IκBα and P-P65) in synovial tissues of knee joints. The results indicated that miR-26a repressed the activation of NF-κB signaling pathway in synovial tissues of knee joints in OA rats. It has been reported that the activation of NF-κB was suppressed by overexpression of miR-26a . In atheBased on observations and evaluations made during the study it was suggested that miR-26a suppressed the activation of the NF-κB signaling pathway in the synovial tissues of knee joints in OA rats, in response to which, the synovial inflammation and cartilage injury in OA rats were alleviated. MiR-26a may act as a novel prognostic marker and new therapeutic target for OA in knee joints. Further work should be performed to obtain more information about the specific mechanisms, and to explore the clinical value for OA.


The qPCR results were further verified by using Western blotting (WB). A total of 10 MCs with different accessions were collected and tested in this study. Among the tested accessions, RU5H showed the highest expression level of the polypeptide-P gene. The expression level of the polypeptide-P gene was not only season-related (with the highest in early July) but also tissue-related (with the highest in the seed tissue). In addition, the expression characteristic of the polypeptide-P gene was maturity-related, with the highest expression level in the tender MC. The WB results show that the transcription level of this gene shows an almost similar trend to the corresponding protein expression level. In conclusion, the established qPCR method can rapidly and effectively detect the expression levels of the polypeptide-P gene in MCs with different accessions; furthermore, various factors, including the accessions, harvesting seasons, and tissue types can affect the expression level.This study was designed to establish a real-time quantitative polymerase chain reaction (qPCR) method to rapidly and reliably analyze the hypoglycemic  Diabetes mellitus (DM), generally referred to as diabetes, is an endocrine disorder with the symptom of persistent high blood glucose levels caused by the decrease or defect of insulin ,2. At prIn general, patients with DM need to keep taking drugs throughout their life, and the DM treatment requires a higher security standard. Thus, it is of great importance to develop novel anti-diabetic drugs with few side effects ,5. Momordica charantia (MC) has been traditionally used for diabetic treatment in Southeast Asia and China due to its antihyperglycemic and antioxidant activities [tivities ,6,7,8,9,tivities ,11. Accutivities ,14,15,16tivities .A previous study showed that the content of polypeptide-P exhibits distinct differences in various MC varieties . Therefopolypeptide-P gene from MC was successfully cloned [polypeptide-P gene, and to identify the key factors affecting the expressed levels in different MC varieties, we established a qPCR procedure in this study to analyze the transcription level of the MC polypeptide-P gene and further analyzed the corresponding protein expression level by using Western blotting (WB). Our results show that the expression level of the polypeptide-P gene in MC is affected by many factors, such as the accessions, harvesting seasons, and tissue types.A real-time quantitative polymerase chain reaction (qPCR) is a potential method for the rapid detection of a target gene expression level and has been widely used in species identification ,21,22,23y cloned . To undeA total of 10 MCs with different accessions were collected in this study . They we260 nm/OD280 nm ratio between 1.9 and 2.1, as well as those with an OD260 nm /OD230 nm ratio greater than 2.0, were used for the analysis. The integrity of RNA samples was also assessed by 1% agarose gel electrophoresis. Subsequently, 1 μg of total RNA was used to synthesize cDNA in a total volume of 20 μL by using the reverse transcriptase , and the 10-fold diluted cDNA was used as a template to quantify the target gene expression level by using qPCR.The total RNA was extracted from each frozen MC sample (approximately 100 mg) based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol . To remow/v). The mixture was stirred continuously for 2 h at room temperature and centrifuged at 6000g for 15 min at 25 °C. The supernatant was collected for further assay.A protocol described previously was used for protein isolation from MC . Brieflyβ-actin was selected as the reference gene. There is no published MC β-actin gene sequence in GenBank, so the partial MC β-actin nucleotide sequence was cloned by designing a pair of primers based on the alignment result of available Cucurbitaceae β-actin gene sequences . The qPCR primers (MC-actin-F and MC-actin-R) for the MC-specific β-actin gene were designed based on the partial MC β-actin nucleotide sequence. The polypeptide-P gene specific qPCR primers were designed based on a previously published gene sequence (accession No. HQ164449) [http://frodo.wi.mit.edu). The MC housekeeping gene Q164449) . The pri2O. The reaction conditions were as follows: an initial denaturation step of 95 °C for 10 min to activate the FastStart Taq DNA polymerase, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Baseline and threshold cycles (Ct) were automatically determined using the StepOne™ System . Biological triplicates of each sample were used for the qPCR analysis. The relative gene expression was calculated based on the 2−ΔΔCt method based on the control gene [The qPCR reactions were performed on 96-well plates with the StepOnePlus™ Real-Time PCR System . The qPCR reaction mixture was prepared in a total volume of 20 μL containing: 2 μL of synthesized cDNA template, 0.8 μL of each amplification primer, 10 μl of 2 × FastStart SYBR Green Master , and 6.4 μL of ddHrol gene .For the WB assay, a total of 100 ng protein, detected via the colloidal Coomassie Brilliant Blue (CBB)  method, was combined with an equal volume of 2 × SDS-PAGE loading buffer and then heated to 100 °C for 8 min and separated by 13.5% Tris-Tricine SDS-PAGE. Proteins were electroblotted onto a PVDF membrane at 100 V for 90 min using a wet blotting system . The electroblotted membranes were blocked with 5% skim milk in PBS containing 0.5% Tween-20 (PBS-T buffer) overnight at 4 °C. After washing the membranes three times with PBS-T buffer, an anti-polypeptide-P antibody , dilutedpolypeptide-P gene were checked for statistical significance according to Student’s t-test. The results were considered statistically significant if the P-value was <0.05. The WB results were analyzed using ImageJ2×  and SPSS 19.0 software .Changes in the relative expression levels of the β-actin has been widely applied in qPCR analysis and is often used as an internal control. The gene alignment result of β-actin from three Cucurbitaceae plants  displays high identity. Therefore, a pair of primers were designed to amplify this highly conserved region of the β-actin gene. The amplified DNA fragment was sequenced, which showed high similarity to the β-actin gene sequences of Cucumis melo , Cucumis sativus , and Citrullus lanatus . As a result, the primers of the MC β-actin were designed according to the cloned DNA sequence information and were used as an internal control for the qPCR analysis . By contrast, the transcription expression levels of the polypeptide-P gene in CL, TDCB, TWDC, and XC2H are lower than that of the control. RU5H displays the highest polypeptide-P gene expression level and XC2H has the lowest. These data indicate that the expression levels of the polypeptide-P gene vary among MCs with different accessions. In China, CB is most widely used as a health food because CB is said to contain a high content of polypeptide-P. Therefore, CB was used as a control sample in the study just to compare the relative expression levels among MCs with different accessions. The qPCR results A show thpolypeptide-P gene expression level.Furthermore, a WB analysis was carried out to examine the expression levels of polypeptide-P in different MC accessions B. The repolypeptide-P gene. polypeptide-P gene on 6 July 6 was found, but after that date, the expression levels significantly declined. These data indicate that the expression level of the polypeptide-P gene is time- or season-dependent and tends to be relatively high in early July, when the MC fruit is mostly immature. To testify this hypothesis, we compared polypeptide-P gene expression levels in the mature fruit with those in the immature fruit. polypeptide-P gene expression levels than the mature fruits, indicating that polypeptide-P gene transcription levels decrease with the maturity and development of fruits.The collection of some MC tissue types was not successful, so we selected four samples  to analyze the impact of harvesting seasons on expression levels of the polypeptide-P gene expression change in different tissues, we systematically analyzed its expression levels in RU5H, which was harvested on 6 July, with the highest expression level of the polypeptide-P gene. The results in polypeptide-P gene in various tissues of RU5H differs greatly. High levels of transcription were observed in the leaf, fruit, and seed of RU5H. Much lower levels of polypeptide-P gene transcription were observed in the stem and flower than in developing seeds and fruits (polypeptide-P gene transcription was found in the developing seed. Thus, the polypeptide-P gene shows a spatially differential expression pattern in RU5H.To examine the d fruits . In partpolypeptide-P gene in MC. In addition, our findings show that the polypeptide-P gene is highly expressed in RU5H and DB compared with other accessions, which was confirmed by the result of the WB analysis. The expression pattern of the polypeptide-P gene was time- and tissue-dependent, and the highest expression level was found in early July, indicating an early regulation of the gene in response to the season. The polypeptide-P gene expression was accumulated in leaf, seed, and fruit, especially immature fruit. The present study therefore reveals that the distribution profiling of polypeptide-P and provides a reliable reference for cultivating and using high-quality hypoglycemic MCs.In summary, the qPCR method established in the study provided a rapid and reliable assay system for detecting the


Therefore, metal divalent cations in the dialysate were suggested to be depleted for the dialysis of low methoxyl pectin.Crude water-extracted pectin (WEP) isolated from creeping fig seeds were mainly fractionated into WEP-0.3 and WEP-0.4 fractions. Fractions were confirmed to be nonstarch, nonreducing sugars, nonpolyphenols and protein-unbounded acidic polysaccharides. Interestingly, a significant difference in solubility was found between WEP-0.3 (higher solubility than WEP) and WEP-0.4 (remarkably insoluble), which was consistent with the amorphous and porous sponge-like structure of WEP-0.3 as well as the crystalline and dense rod-like state of WEP-0.4. However, the result of the FT-IR spectra was contradicted by the solubility of WEP-0.4, which possessed the lowest degree of methoxylation and ought to possess the highest solubility. Through mineral analysis, a considerably high content of Ca It has been extensively applied to the food, cosmetic and pharmaceutical industries owing to its unique functional properties of gelling, emulsifying and stabilizing ,2. The scks, RG) ,4. Commocks, RG) ,6. It iscks, RG) . Howevercks, RG) . Meanwhicks, RG) , alkali cks, RG) , pectin cks, RG)  and ammocks, RG) . A betteFicus pumila Linn) of the Moraceae family is a perennial and scandent shrub plant that grows vigorously on mountain sides and broken walls, as well as along the trunks of tall trees in China, Japan and some other countries. The seeds of this plant, which are rubbed and squeezed in water to yield a mucilaginous water extract, have a long history of being used to make a summer drink named “Liang fen” for local people. In our previous study, the main component of the water extract was found to be LMP. This pectin has the peculiarity of being extracted more easily when compared with other industrial pectin resources, such as citrus peel and apple pomace, because it is located in a transparent layer on the surface of seeds [Creeping fig  isolated from creeping fig seeds was fractionated into WEP-0.3 and WEP-0.4 by anion-exchange chromatography. It was surprising to find that these two fractions exhibited a significant difference in solubility. Therefore, a series of experiments, including environmental scanning electron microscopy (ESEM), X-ray diffraction, FT-IR spectroscopy and mineral analysis were carried out to explore the reasons for the solubility difference.Galacturonic acid was procured from Sigma Aldrich Co., Shanghai, China. Bovine serum albumin was supplied by Bioleaf Biotech Co. Ltd. . Coomassie brilliant blue G-250 was procured from Solarbio Biotech Co. Ltd. . All chemical reagents used in the experiments were analytical grade.w/v) was applied. The extracts were then filtered to remove the solid sediments and precipitated by the addition of ethanol to obtain a final concentration of 50% (v/v). Thereafter, the precipitates were washed successively with ethanol/water mixtures of the following volume ratios: 70/30, 80/20, 90/10, as well as absolute ethanol. The obtained pectins were lyophilized and stored in hermetically sealed glass bottles.The preparation of plant materials and extraction of WEP were carried out according to our previous method . BrieflyThe fractionation of WEP was performed by using anion-exchange chromatography according to the method of Sun et al.  with som3 reaction, carbazole reaction, full wavelength scanning and Coomassie brilliant blue reaction [General physicochemical properties were assayed by using the following methods: color observation, iodination reaction, Fehling’s test, FeClreaction .i is defined as initial weight of the samples, and WS is the weight of the dried insoluble pectin portions.The water solubility of WEP and its fractions was determined by the method of Monsoor et al. . SamplesThe X-ray diffraction patterns of samples were recorded with an X-ray diffractometer  operated at 40 kV and 40 mA with Cu Kα radiation. The samples were scanned over the range of 5° to 60° diffraction angle (2θ) with a step size of 0.02° (2θ) and a counting time of 0.2 s/step.Samples were mounted onto a circular specimen stub using double-sided tape and observed using an environmental scanning electron microscope (ESEM)  at 30 kV voltage with a 3.0 spot size. Low vacuum mode was used while operating the ESEM.−1 [−1) and analyzed with Ominic 7.2 software .The FT-IR spectra of pectin were recorded on a Nicolet 5700 spectrometer . The dried samples were ground with spectroscopic-grade KBr powder  and pressed into pellets for spectra measurement in the frequency range of 4000–400 cm−1 . The dat−1 (υ(C=O)COOMe) over the sum of the peak areas at 1740 cm−1 and 1630 cm−1 (υas (COO)) based on pectin standards with known DMs. After that, the peak area of the ester carbonyl of sample was fit to the curve to calculate its DM.The degree of methoxylation (DM) was calculated by using FT-IR spectra based on the method of Gnanasambandam et al. . A lineaMineral composition and content were determined by using inductively coupled plasma–atomic emission spectrometry (ICP–AES) . About 1 gram of pectin was accurately weighed into a 100-mL beaker, and 20 mL mixed acid containing nitric acid/perchloric acid (3:1) was added. The sample was heated on a heating plate for 40 min, and then quantitatively transferred to a volumetric flask with a fixed capacity of 100 mL. The supernatant was used for the mineral analysis.All the experiments were conducted in triplicate. Statistical analysis was carried out using SPSS . The results are expressed as means ± standard deviations and were compared using the Tukey test at a 5% confidence level.The charge heterogeneity of WEP can be observed from the elution curve of the anion-exchange column . Two majPectin is usually provided in a powdery form and must be dissolved in an aqueous solution prior to use. Its solubility and dissolution kinetics are thus pivotal specifications in a large number of technological application processes. 3 reaction were presented, thus revealing the absence of starch, reducing sugars and polyphenol in WEP and its fractions. Coomassie Brilliant blue assays suggested that protein was present in WEP. Nevertheless, no protein was found in WEP-0.3 and WEP-0.4 fractions. This result implied that the protein in WEP was unbounded, which could be detached after fractionation. The full wavelength scan also confirmed that protein was not present in WEP-0.3 and WEP-0.4 fractions. Therefore, the solubility difference between WEP-0.3 and WEP-0.4 was not attributable to the presence of insoluble materials, but due to other reasons.A general physicochemical properties analysis was performed to check the possibility of the presence of insoluble materials in WEP and its fractions and the results are listed in It was reported that amorphous solids generally have higher solubility and higher dissolution rate because they have higher free energy than corresponding crystals . TherefoThe significant difference in solubility of WEP and its fractions may also relate to the morphology of pectin. As shown in −1 and 1627 cm−1, which separately featured the presence of a methyl esterified carboxylic group and a free carboxylic group. The ratio of the peak area at 1740 cm−1 over the sum of the peak areas at 1740 and 1620 cm−1 is often used to calculate the DM [2+, causing WEP-0.4 to have difficulty in dissolving. Therefore, the next step was to determine the mineral content of WEP and its fractions in order to validate the deduction.Properties of pectin such as solubility, crystalline structure and microscopic morphology may relate to the molecular structure. In order to further demonstrate the difference in solubility of WEP and its fractions, their FT-IR spectra were investigated. As e the DM . Hence, 2+ was detected in water extracts from creeping fig seeds without precipitation and washing [2+ in WEP was still high after successive precipitation and washing. The redundant Ca2+ was probably derived from the high Ca2+ in seeds, and tap water with a high Ca2+ content was used as dialysate during the initial stage. In comparison with significantly reduced level of magnesium, the levels of calcium were increased after dialysis. The content of Ca2+ in WEP-0.4 was significantly higher than that in WEP and WEP-0.3, indicating that WEP-0.4 had stronger binding abilities towards calcium. The low solubility of WEP-0.4 may be the consequence of microgel formation in the presence of a high content of Ca2+ and a lower DM. Hence, dialysate which contains multivalent metal ions such as Ca2+ should be avoided when LMP is dialyzed. washing . HoweverIn the present work, two main fractions (WEP-0.3 and WEP-0.4) of crude water-extracted pectin (WEP) isolated from creeping fig seeds were obtained by fractionation using anion-exchange chromatography. The general physicochemical properties of WEP and its fractions were assayed and a significant difference in solubility of the two fractions was noticed. WEP-0.3 possessed a higher solubility than WEP, while WEP-0.4 became distinctly insoluble. An endeavor was made to explain the solubility difference of WEP-0.3 and WEP-0.4 from the point of crystalline structure, microscopic morphology and molecule structure. Nevertheless, the FT-IR spectra showed that WEP-0.4 had the lowest DM, which should lead to the highest solubility. The low solubility of WEP-0.4 was probably attributed to the considerably high calcium content and the lower DM of WEP-0.4 that was prone to forming microgels during dialysis. Therefore, a solution containing a metal divalent cationic was suggested to be forbidden for the dialysis of low methoxyl pectin.


Acinetobacter baumannii (Ab) is one of the most important pathogens associated with nosocomial infections, especially pneumonia. Interest in the Quorum network, i.e., Quorum Sensing (QS)/Quorum Quenching (QQ), in this pathogen has grown in recent years. The Quorum network plays an important role in regulating diverse virulence factors such as surface motility and bacterial competition through the type VI secretion system (T6SS), which is associated with bacterial invasiveness. In the present study, we investigated 30 clinical strains of A. baumannii isolated in the “II Spanish Study of A. baumannii GEIH-REIPI 2000-2010” (Genbank Umbrella Bioproject PRJNA422585), a multicentre study describing the relationship between the Quorum network in A. baumannii and the development of pneumonia and associated bacteraemia. Expression of the aidA gene  was lower (P < 0.001) in strains of A. baumannii isolated from patients with bacteraemic pneumonia than in strains isolated from patients with non-bacteraemic pneumonia. Moreover, aidA expression in the first type of strain was not regulated in the presence of environmental stress factors such as the 3-oxo-C12-HSL molecule  or H2O2 . However, in the A. baumannii strains isolated from patients with non-bacteraemic pneumonia, aidA gene expression was regulated by stressors such as 3-oxo-C12-HSL and H2O2. In an in vivo Galleria mellonella model of A. baumannii infection, the A. baumannii ATCC 17978 strain was associated with higher mortality (100% at 24 h) than the mutant, abaI-deficient, strain (carrying a synthetase enzyme of Acyl homoserine lactone molecules) (70% at 24 h). These data suggest that the QS (abaR and abaI genes)/QQ (aidA gene) network affects the development of secondary bacteraemia in pneumonia patients and also the virulence of A. baumannii. Acinetobacter baumannii is a major cause of hospital-acquired infections associated with high mortality rates   high genetic versatility, facilitating rapid adaptation to stressful or unfavorable situations , an enzyme that synthesizes signaling molecules (AbaI) and a receptor protein activator of the QS (AbaR), which forms a complex with N-(3-hydroxydodecanoil)-L-homoserine lactone (3-OH-C12-HSL) to regulate virulence factors, biofilm formation, surface motility, and bacterial competence (T6SS)  in the respiratory tract  network is generally used by Gram-negative bacterial pathogens to regulate biological processes such as virulence, conjugation, resistance, biofilm formation  and functionally characterized in clinical strains of A. baumannii capable of inhibiting their own QS (by Quorum Quenching)   has recently been cloned in abaR (QS) and aidA (QQ) genes and the development of pneumonia and bacteraemia in clinical strains of A. baumannii isolated in the “II Spanish Study of A. baumannii GEIH-REIPI 2000-2010,” a multicentre study involving 45 Spanish hospitals and 246 patients. In addition, we used an in vivo infection model consisting of larvae of the wax moth Galleria mellonella to examine the relationship between the global QS/QQ and the development of mortality by a mutant abaI (QS)-deficient strain of A. baumannii (A. baumannii ATCC17978ΔabaI) relative to that of the wild-type A. baumannii ATCC17978 strain.Based on these findings, in the present study, we examined the relationship between the global Quorum regulatory network (QS/QQ) mediated by the A. baumannii from the 465 strains isolated in the “II Spanish Study of A. baumannii GEIH-REIPI 2000-2010” multicentre study (Genbank Umbrella Bioproject PRJNA422585). The multicentre study included 45 hospitals in Spain, in which new cases of colonization or infection by A. baumannii were analyzed between February and March 2010  or A. baumannii colonization of the lower respiratory tract (n = 17) , non-bacteraemic pneumonia (Pn-NB)  and the QQ enzyme (aidA) in A. baumannii ATCC 17978 (Genbank genome accession numbers CP000521.1 [CP018664.1]) and in 1000 A. baumannii genomes by consulting the “Integrated Microbial Genomes and Microbiomes” web page (https://img.jgi.doe.gov) and using nucleotide BLAST. The gene sequences used in the search were selected from the Acinetobacter baumannii ATCC 17978 genome. A threshold of 1e-50 was used as the limit for analysis of the nucleotide sequence, where the e-value was defined as the probability of random alignments with the same score. We also calculated the percentage presence of these genes in the genomes  plates and incubated at 37° C for 24 h. One colony was removed and inoculated in liquid LB medium and incubated overnight at 37° C under stirring at 180 rpm. The inoculum was diluted (1:100) and allowed to grow until an optical density (OD600 nm) of 0.4–0.6 (corresponding to the logarithmic growth phase) was reached. The RNA was then extracted using the High Pure RNA Isolation kit  and the extract was treated with Dnase . The extracted RNA was subsequently quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and the concentration was adjusted to 50 ng/μl in order to yield efficiencies of 90-110%  plates and incubated at 37°C for 24 h. One colony was then removed, inoculated in liquid LB medium and incubated overnight at 37°C under stirring at 180 rpm. The preinoculum was diluted (1:100) and allowed to grow until an optical density (OD600 nm) of 0.3 was reached. Aliquots of 10 μM of 3-Oxo-C12-HSL (QS-inactivating molecule by expression of the AidA protein)  , as well as 5 min under H2O2 in static at 37°C, RNA was extracted using the High Pure RNA Isolation kit  and treated with Dnase. The extracted RNA was subsequently quantified as described above , under the following conditions: reverse transcription at 63°C for 3 min, denaturation at 95°C for 30 s, followed by 45 cycles of 15 s at 95°C and 45 s at 60°C and, finally, cooling at 40°C for 30 s. The UPL primers and probes from conserved DNA regions identified by PCR  used in the analysis are shown in Table rpoB, for RT-qPCR studies of Quorum sensing Primer sequences (5′-3′) with Taqman probes . Each experiment was carried out in duplicate with two RNA extracts (50 ng/μl). For each strain, the expression of all genes, primers, and probes was normalized relative to the reference or housekeeping gene, Galleria mellonella model was an adapted version of that developed by Peleg et al. , were each injected with 10 μl of a suspension of A. baumannii ATCC17978, or its isogenic deficient mutant A. baumannii ATCC17978ΔabaI, diluted in sterile phosphate buffer saline (PBS) and containing 8 × 104 CFU (± 0. 5 log). The injection was performed with a Hamilton syringe (volume 100 μl) . In addition, a control group of twelve larvae were injected with 10 μl of sterile PBS. After being injected, the groups of larvae were placed in Petri dishes and incubated in darkness at 37°C. The number of dead larvae was recorded twice a day (morning and afternoon) for 6 days. The larvae were considered dead when they showed no movement in response to touch . The graphs were constructed using the GraphPad program, and the results were represented as means and their respective standard deviations.The gene expression studies were carried out in duplicate, and the data obtained were analyzed by Student's in vivo Galleria mellonella infection model were constructed using GraphPad Prism v.6 and the data were analyzed using the Log-rank test (Mantel-Cox). In both cases, p-values < 0.05 were considered statistically significant, and the data were expressed as mean values.The mortality curves corresponding to the A. baumannii clinical strains .The statistical analyses were applied to the following categorical variables: age, sex, immunosuppressive treatment, surgery, ICU stay, mechanical ventilation, tracheostomy, severe sepsis, septic shock, and expression of the Quorum genes in abaR and aidA genes of the Quorum network (QS/QQ) was quantified by RT-qPCR analysis of the 17 isolates of A. baumannii from colonized patients and of the 13 isolates of A. baumannii from patients with pneumonia  of the a Figure . The meaabaR, aidA) between clinical strains of A. baumannii isolated from colonized patients and strains of A. baumannii isolated from patients with pneumonia .The results did not reveal any significant differences in the RE of the Quorum network genes  from those isolated from patients without bacteraemia (Pn-NB). The resulting graphs are shown below  and those with non-bacteraemic pneumonia (Pn-NB). We observed that abaR gene was overexpressed in A. baumannii isolates from Pn-B patients relative to Pn-NB patients . By contrast, the aidA gene was overexpressed in A. baumannii clinical strains in Pn-NB patients relative to Pn-B patients   than that of isolates from Pn-NB patients.We then proceeded to study the RE of the w Figure . The fin) Figure . Only onabaR and aidA genes (Quorum network) in the presence of 3-Oxo-C12-HSL (Inhibition of the QS) and H2O2 (Activation of the QS), obtained by RT-qPCR of the 13 isolates of A. baumannii from patients with pneumonia (differentiated from Pn-NB) are shown in Table abaR/aidA (QS/QQ) genes between the different clinical isolates   Figure  and of H5 Figure ]. ExpresA. baumannii isolated from Pn-B , in contrast to the isolates of A. baumannii from Pn-B, which did not have this functional protein. Therefore, in the A. baumannii strains isolated from Pn-B, overexpression of the abaR gene (activation of the QS) in the presence of H2O2 (ROS response) would enable the development of the virulence factors favoring invasiveness, such as type VI secretion system (T6SS) and motility.These results indicate that the isolates of A. baumannii revealed only one statistically significant variable, i.e., diabetes mellitus   caused 100% mortality after 24 h, whereas injection of the larvae with the same concentration of A. baumannii ATCC17978ΔabaI resulted in 70% mortality after 24 h .Injection of A. baumannii, abaR and abaI (QS system) and aidA (QQ mechanism) in relation to clinical features of pneumonia and bacteraemia. Although other QQ enzymes have been described in A. baumannii ATCC 17978  genes that differed between genomes of clinical isolates of A. baumannii isolated from patients with bacteraemic pneumonia (Pn-B), the abaR gene was overexpressed (p < 0.05). The AbaR protein was the receptor activator of the Quorum Sensing system (QS), and the aidA gene was not expressed. Moreover, we observed regulation of aidA gene expression in clinical strains of pneumonia-causing A. baumannii  by the 3-Oxo-C12-HSL molecule (which is an AidA enzyme substrate in QQ activity) and H2O2 (an activator of the QS system). However, there was no difference in the expression of Quorum network genes between colonized and pneumonia patients, as previously described , underexpression of the aidA gene was also the only statistically significant variable (P < 0.05).On the other hand, clinical analysis of the risk factors associated with pneumonia caused by Yersinia pseudotuberculosis, Proteus mirabilis, and Vibrio cholerae, the QS system is the main regulatory mechanism of bacterial competence via T6SS, which is involved in the invasiveness and motility that favor the development of bacteraemia   isolated from blood and which was the origin of a bacteraemic outbreak  in various virulence factors such as biofilm formation and motility. In both cases, abaI deficiency led to a decrease in biofilm production and motility , similar effects have been observed in other studies, in which injection of G. mellonella larvae with the reference strain A. baumannii ATCC17978 resulted in rapid death. Mortality was significantly dependent on the number of cells injected. More than 75% of the larvae died in the first 48 h of injection with at least 3.7 × 105 CFU / larva, while very few of the larvae died after being injected with a concentration equal to or lower than 3.7 × 104 CFU/larva (p < 0.01)  was obtained, demonstrating that inactivation of both QS systems leads to a significant reduction in pathogenicity (p < 0.01) when virulence factors are not activated, such as the type VI secretion system (T6SS) and motility /QQ (aidA gene) network plays a role in the development of bacteraemia in patients with pneumonia caused by A. baumannii. This is the first study reporting a relationship between reduced expression of this bacterial QQ enzyme gene (AidA protein) and bacteraemia. Further studies of this relationship in the same and other bacterial QQ enzymes would be of great interest.In conclusion, our findings suggest that the QS (LF-G, AA, LB, IB, ML and RA-M developed the experiments. FF-C, LM-M, JV, JR-B, JG-M, JMC, AP, JP, GB, and YS wrote the manuscript and provided the strains. MT led the experiments and manuscript redaction.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.